PROJECT SUMMARY The primary goal of our research is to establish whether the pyruvate dehydrogenase kinases (PDKs) should be considered therapeutic targets for the treatment of hepatic steatosis, obesity, and type 2 diabetes. The long-term objective is to find inhibitors of the PDKs that will lead to development of effective drugs for the treatment of these conditions. The immediate objective is to perform proof-of- principle studies with PDK knockout mice generated in my laboratory. Two lines of evidence support our hypothesis that the PDKs are viable targets for the treatment of these conditions. First, down regulation of the expression of PDKs is a critical component of the mechanism by which insulin controls blood glucose. Second, PDK knock out mice maintained on a high-fat, diabetogenic diet accumulate less fat in their livers, are less obese, and have lower blood glucose levels, better glucose tolerance, and greater insulin sensitivity than wild type mice. The activity of the pyruvate dehydrogenase complex (PDC) is tightly regulated by phosphorylation by the PDKs. The phosphorylation state of PDC is controlled by changes in expression level of the PDKs. PDC is inactivated in starvation and diabetes because of induction of PDK2 and 4 by high levels of glucocorticoids and fatty acids and low levels of insulin. That glucose homeostasis depends upon regulation of PDC, which in turn depends upon hormonal control of the amounts of the PDKs, was the original hypothesis for this work. Studies with knockout mice for PDK2 and PDK4 have confirmed this hypothesis. Preliminary studies indicate that knocking out both PDK2 and PDK4 in the same mouse results in an even greater improvement in glucose tolerance and insulin sensitivity than knocking out PDK4 alone. The specific objectives of the proposal are: (a) determine the effects that knocking out both PDK2 and PDK4 in the same mouse has upon fasting blood glucose levels, glucose tolerance, insulin sensitivity, hepatic steatosis, adiposity, branched chain amino acids (BCAAs), free fatty acids (FFAs), ketone bodies, triacylglcyerols (TAG), cholesterol, blood pH, and markers of inflammation and oxidative stress in mice fed a high saturated fat diet; (b) determine the molecular mechanisms responsible for the increases in blood levels of ketone bodies in PDK knockout mice; and (c) determine whether knocking out PDK2 and PDK4 inhibits glyceroneogenesis by reducing the availability of lactate, pyruvate, and alanine. Following completion of these proof-of-principle studies, we hope to shift our goal to the identification of small molecule inhibitors of PDKs for the treatment of hepatic steatosis, obesity, and type 2 diabetes. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Hepatic steatosis, the accumulation of fat in the liver, is a common feature of nonalcoholic fatty liver disease (NAFLD) that occurs in 14 to 20% of the US population [63]. Once considered benign, NAFLD is now known to be a risk factor for nonalcoholic steatohepatitis (NASH), a serious condition in which inflammation kills liver cells, ultimately culminating in liver cirrhosis, portal hypertension, and liver failure. Although NASH is less prevalent (about a quarter of the population with NAFLD have NASH), the number is growing in parallel with the increase in NAFLD. 27,000 Americans die each year from cirrhosis. Hepatic steatosis, obesity, insulin resistance, and type 2 diabetes mellitus are all closely linked [64]. Indeed the majority of obese, type 2 diabetic patients also have hepatic steatosis. Since a fourth of patients treated at Veteran Administration hospitals have type 2 diabetes [65], many veterans have NAFLD and are at risk of developing NASH and dying of cirrhosis. Furthermore, veterans exposed to dioxin- contaminated herbicides in Vietnam are particularly prone to the development of type 2 diabetes and therefore hepatic steatosis, NASH, and cirrhosis [66]. Learning how to prevent NAFLD and its progression to NASH depends upon a better understanding of why fat accumulates in tissues and why the amount of fat that accumulates usually correlates with the degree of insulin resistance. Fatty acids synthesized in the liver in the absorptive state should be esterified to glycerol and shipped to the adipose tissue for storage, never retained in the liver. Fatty acids delivered to the liver from the adipose tissue in the postabsorptive state should be either oxidized or re-esterified to glycerol and shipped back to the adipose tissue. The reason why fatty acids are retained in the liver as TAG in NAFLD is poorly understood. Part of what needs further clarification is the role of PDC in regulation of relative rates of fatty acid and glucose oxidation. A better understanding of how regulation of PDC affects fat accumulation and insulin sensitivity of the liver could help in the effort to develop pharmaceuticals for the prevention of NAFLD and NASH. Our hope is that work on this enzyme complex will lead to the development of novel drugs for the treatment of hepatic steatosis, obesity, and type 2 diabetes. We therefore believe the proposed work has the potential to have a significant impact on the health care of Veterans.